1. Field of the Invention
The present invention relates generally to the fields of drug delivery. More particularly, it concerns methods for preparing and administering a protein-stabilized lipid formulation containing at least one pharmaceutical agent.
2. Description of Related Art
Liposomes are phospholipid vesicles, composed mainly of naturally occurring substances that are nontoxic and biodegradable (Lasic 1993). They are made up of at least one lipid bilayer membrane containing an entrapped aqueous internal compartment. When combined with water, phospholipids immediately form a sphere because one end of each molecule is water soluble, while the opposite end is water insoluble. Water-soluble medications added to the water are trapped inside the aggregation of the hydrophobic ends; fat-soluble medications are incorporated into the phospholipid layer.
Liposomes are particularly useful for drug delivery. Liposomes have been employed for a number of therapeutic applications, in particular, for delivering drugs to target cells following systemic administration (Drummond et al., 1999; Gibbson and Paphadjopoulos 1988; Lasic and Paphadjopoulos 1998; Olson et al., 1982; Rahman et al., 1982; Working et al., 1994a; Working et al., 1994b; Working et al., 1996; Working et al., 1999; Mayer et al., 1989). Liposomal formulations of pharmaceutical agents are often superior to drugs injected in the free form. When used in the delivery of certain cancer drugs, liposomes help to shield healthy cells from the drugs' toxicity and prevent their concentration in vulnerable tissues (e.g., the kidneys, and liver), lessening or eliminating the common side effects of nausea, fatigue, and hair loss. For instance, liposomal formulations of the anticancer agent vincristine exhibit greater efficacy against L1210 leukemia cells than does free vincristine and have reduced collateral toxicity. Liposomes have also been used to deliver certain vaccines, enzymes, or insulin to the body. They have also been used experimentally to carry normal genes into a cell in order to replace defective, disease-causing genes.
Commercial liposomal drug delivery is gaining attention because of the enhanced stability of liposomes, reduced toxicity, improved pharmacokinetics, enhanced blood circulation time, and increased accumulation of liposomes in the target sites. Reduction in toxicity may result from the ability of liposomes to decrease drug exposure, and subsequent damage, to susceptible tissues (Allen et al. 1991). In fact, the first liposomal drug oncology formalations approved for medicinal use, were of the anthracyclines daunorubicin (DaunoXome; Nextstar Pharmaceuticals, Boulder, Colo.), EVACET (The Liposome Company, Inc., Princeton, N.J.) and DOX [Doxil; Alza Corporation, Palo Alto, Calif. (CAELYX in Europe)].
The use of liposomes as a vehicle of drug delivery has produced many promising results. Major advances in improving the therapeutic index of amphotericin B encapsulated in liposomes have been demonstrated in counteracting systemic fungal infections in cancer patients (Olsen et al. 1982). The liposomal entrapment of this antifungal drug causes a remarkable reduction in toxicity. Liposomes have also been found to be effective in delivering doxorubicin (Williams et al. 1993), vincristine (Woodle et al. 1992), vinblastine, actinomycin-D, arabinoside, cytosine, daunomycin (Julliano and Stamp 1978), mitoxantrone, epirubicin, daunorubicin, (Madden et al 1990) and paclitaxel (Suffness 1995). In a liposomal drug delivery system, if the drug is highly hydrophobic, it tends to associate mainly with the bilayer compartment (Sharma et al., 1995, 1997).
Several methods have been utilized for the production of liposomes. Because vesiculation of natural phospholipid bilayers is not a spontaneous process, physical and chemical methods are used to produce well-defined liposomes from hydrated lipids. Usually, these methods require the input of high energy (e.g., ultrasonic treatment, high pressure, and/or elevated temperatures) to disperse low critical micelle concentration phospholipids as a metastable liposome phase (Lasic and Paphadjopoulos 1998).
One method for the preparation of liposomes involves the solvent evaporation of an oil-in-water emulsion. The oil-phase contains one or more pharmaceutical agents, cholesterol and lipids and the aqueous phase contains an emulsifier. An emulsion consists of two immiscible liquids (usually oil and water), with one of the liquids dispersed as small spherical droplets in the other.
In addition to having utility for drug delivery, liposome-like emulsions can be found in several foods, such as mayonnaise, milk, margarine, and butter. In most foods that contain emulsions of oil and water, the diameters of the droplets usually lie somewhere between 0.1 and 100 μm (Dickinson and Stainsby, 1982, Dickinson, 1992). An emulsion can be conveniently classified according to the distribution of the oil and aqueous phases. A system that consists of oil droplets dispersed in an aqueous phase is called an oil-in-water or O/W emulsion (e.g., mayonnaise, milk, cream etc.). A system that consists of water droplets dispersed in an oil phase is called a water-in-oil or W/O emulsion (e.g., margarine, butter and spreads). The process of converting two separate immiscible liquids into an emulsion, or of reducing the size of the droplets in a preexisting emulsion, is known as homogenization.
In both liposomes used for drug delivery and emulsions in food products, the breakdown of the vesicle structure of the compositions has been observed. The term “emulsion stability” is broadly used to describe the ability of an emulsion to resist changes in its properties with time. Emulsions may become unstable through a variety of physical processes including creaming, sedimentation, flocculation, coalescence, and phase inversion. Creaming and sedimentation are both forms of gravitational separation. Creaming describes the upward movement of droplets due to the fact that they have a lower density than the surrounding liquid, whereas sedimentation describes the downward movement of droplets due to the fact that they have a higher density than the surrounding liquid. Flocculation and coalescence are both types of droplet aggregation. Flocculation occurs when two or more droplets come together to form an aggregate in which the droplets retain their individual integrity, whereas coalescence is the process where two or more droplets merge together to form a single larger droplet. Extensive droplet coalescence can eventually lead to the formation of a separate layer of oil on top of a sample, which is known as “oiling off”.
Thermodynamics are largely responsible for the separation of phases. If an emulsion is generated by homogenizing pure oil and pure water together, the two phases will rapidly separate into a system that consists of a layer of oil (lower density) on top of a layer of water (higher density). This is because droplets tend to merge with their neighbors, which eventually leads to complete phase separation.
The disruption of liposome structure over time and premature drug leakage present significant, and potentially very hazardous, problems for using liposomes as vehicles for drug delivery. Drug leakage from liposomes during long-term storage, lyophilization and reconstitution can decrease the predictability and increase the toxicity of drug delivery using liposomes. Specifically, the premature release and leakage of the drug from the liposome results in a faster distribution of the drug in the plasma component, increased toxicity, and decreased concentrations of the drug released at the tumor site. Furthermore, for pegylated liposomal doxorubicin, a novel dose-limiting form of skin toxicity known as palmar-plantar erythrodysaesthesia or hand-foot syndrome has been described (Gordon et al. 1995). Thus a need exists to improve liposome design to increase liposome stability and eliminate premature drug leakage.
In the food industry, the use of emulsifiers and/or thickening agents have been used to produce more stable emulsions. Emulsions usually are thermodynamically unstable systems. It is possible to form emulsions that are kinetically stable (metastable) for a reasonable period of time (a few minutes, hours, days, weeks, months, or years) by including substances known as emulsifiers and/or thickening agent prior to homogenization.
Emulsifiers are surface-active molecules that adsorb to the surface of freshly formed droplets during homogenization, forming a protective membrane that prevents the droplets from coming close enough together to aggregate. Most emulsifiers are molecules having polar and nonpolar regions in the same molecule. The most common emulsifiers used in the food industry are amphiphilic proteins, small-molecule surfactants, and monoglycerides, such as sucrose esters of fatty acids, citric acid esters of monodiglycerides, salts of fatty acids, etc (Krog, 1990).
Thickening agents are ingredients that are used to increase the viscosity of the continuous phase of emulsions and they enhance emulsion stability by retarding the movement of the droplets. A stabilizer is any ingredient that can be used to enhance the stability of an emulsion and may therefore be either an emulsifier or thickening agent.
An efficient emulsifier produces an emulsion in which there is no visible separation of the oil and water phases over time. Phase separation may not become visible to the human eye for a long time, even though some emulsion breakdown has occurred. A more quantitative method of determining emulsifier efficiency is to measure the change in the particle size distribution of an emulsion with time. An efficient emulsifier produces emulsions in which the particle size distribution does not change over time, whereas a poor emulsifier produces emulsions in which the particle size increases due to coalescence and/or flocculation. The kinetics of emulsion stability can be established by measuring the rate at which the particle size increases with time.
In oil-in-water emulsions, proteins are used mostly as surface active agents and emulsifiers. One of the food proteins used in o/w emulsions is whey proteins. The whey proteins include four proteins: β-lactoglobulin, α-lactalbumin, bovine serum albumin and immunoglobulin (Tomberg, 1990). Commercially, whey protein isolates (WPI) with isolectric point ˜5 (Tomberg, 1990) are used for o/w emulsion preparation. According to Hunt (1995), whey protein concentrations of 8% have been used to produce self-supporting gels. Later on, the limiting concentrations of whey protein to produce self-supporting gels are known to be reduced to 4–5%. It is possible to produce gels at whey protein concentrations as low as 2% w/w, using heat treatments at 90° C. or 121° C. and ionic strength in excess of 50 mM (Hunt et al, 1995).
Proteins derived from whey are widely used as emulsifiers in the food industry (Phillips et al, 1994; Dalgleish, 1996). They adsorb to the surface of oil droplets during homogenization and form a protective membrane, which prevents droplets from coalescing. The physicochemical properties of emulsions stabilized by whey protein isolates (WPI) are related to the aqueous phase composition (e.g, ionic strength and pH) and the processing and storage conditions of the product (e.g, heating, cooling, and mechanical agitation). Emulsions are prone to flocculation around the isoelectric point of the WPI, but are stable at higher or lower pH (Philips et al, 1994). The resistance to flocculation may be interpreted in terms of colloidal interactions between droplets, i.e, van der Waals, electrostatic repulsion and steric forces (Philips et al, 1994; Dalgleish, 1996). The van der Waals interactions are fairly long-range. Electrostatic interactions between similarly charged droplets are repulsive, and their magnitude and range decrease with increasing ionic strength. Short range interactions become important at droplet separations of the order of the thickness of the interfacial layer or less (e.g., steric, thermal fluctuation and hydration forces; Israelachvili, 1992). Such interactions are negligible at distances greater than the thickness of the interfacial layer, but become strongly repulsive when the layers overlap, preventing droplets from getting closer.
Although emulsifiers and thickening agents have been successfully used in the food industry, there is still a significant need for improving drug-delivering liposome stability. Because most small-molecule chemotherapeutic agents have widespread distribution via i.v. administration (Chanber and Longo, 1996; Speth et al., 1988), this results in a narrow therapeutic index due to a high level of toxicity in healthy tissues. A method for producing more stable liposomes would result in more consistent pharmacokinetics while retaining the advantages of liposomal delivery.